CN109512821B - Crizotinib and 17-AAG composite polymer nano micelle injection, preparation method and application thereof - Google Patents
Crizotinib and 17-AAG composite polymer nano micelle injection, preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a crizotinib and 17-AAG composite polymer nano micelle injection with a synergistic effect, a preparation method thereof and application thereof in preparing a medicament for treating drug-resistant ALK positive non-small cell lung cancer. The preparation comprises 1 part by weight of crizotinib; 0.5-3 parts by weight of 17-AAG; 10-100 parts of nano micelle polymer. The crizotinib and 17-AAG composite nano micelle is uniform in particle size, and the average particle size is 20-200 nm. The preparation can greatly increase the water solubility and the stability of the preparation, prolong the circulation time of the crizotinib and 17-AAG in blood, overcome the condition that a polymer nano-micelle preparation taking polyethylene glycol (PEG) as a shell is not suitable (for example, treating a patient with PEG antibody or a patient who produces anti-PEG antibody after multiple times of administration), passively target a tumor part by enhancing the penetration and retention effect (EPR effect), effectively inhibit the crizotinib-resistant ALK positive non-small cell lung cancer cells, and has the synergistic effect of drug combination.
Description
Technical Field
The invention belongs to the field of pharmaceutical preparations, and relates to a crizotinib and 17-AAG composite polymer nano micelle injection, a preparation method and application thereof in a medicine for treating drug-resistant ALK positive non-small cell lung cancer.
Background
Lung cancer is one of the most prevalent cancers worldwide, and non-small cell lung cancer (NSCLC) accounts for 80% -85% of the total number of lung cancers. Preliminary epidemiological studies have shown that the Anaplastic Lymphoma Kinase (ALK) positivity rate is approximately 3% to 5% in NSCLC patients, which means that approximately 6500-11000 ALK-positive NSCLC patients are diagnosed in the united states annually, and 40000 patients are globally estimated to be ALK-positive NSCLC annually. Crizotinib (Crizotinib) is a small-molecule tyrosine kinase inhibitor developed for ALK-positive NSCLC, is designed according to a eutectic structure of PHA-665752 and non-phosphorylated MET kinase domain, can inhibit phosphorylation of ALK and c-Met of cells so as to inhibit activity of c-Met/Hepatocyte Growth Factor Receptor (HGFR), ALK and oncogenic variants thereof (such as c-Met/HGFR mutants or ALK fusion proteins), further block signal pathways and downstream signals of the ALK, and show strong selective growth inhibition and tumor cell apoptosis promotion activity on tumor cells with c-Met/HGFR gene locus amplification or ALK gene locus translocation/inversion (such as EML4-ALK or NPM-ALK fusion variants). In the clinical studies carried out, ALK-positive NSCLC was selected as the study population based on biomarker detection, and significantly better Objective Remission Rate (ORR) and progression-free survival time (PFS) were observed than in previous chemotherapy. FDA approved for ALK-positive locally advanced or metastatic non-small cell lung cancer in 2011, and FDA approved crizotinib for treatment of advanced ROS 1-positive non-small cell lung cancer patients in 2016. At present, crizotinib is the best choice for the first-line treatment of EML4-ALK fusion gene positive advanced lung cancer patients.
Although the benefits of ALK-positive lung cancer patients are significant, this group of patients often develop resistance to crizotinib within 1-2 years, and recurrent progression of the central nervous system is more common. The drug resistance mechanism is various and can be divided into two main categories: ALK resistance mutations and other signaling pathway switches (i.e., activation of signaling pathways). Therefore, the targeted development of a novel preparation capable of resisting crizotinib drug resistance can greatly meet the requirement of clinical treatment of ALK positive non-small cell lung cancer. Heat shock protein 90(HSP90) inhibitors were found to be active on ALK-positive, non-crizotinib-treated or crizotinib-resistant cell lines in vitro cell line studies. The HSP90 inhibitor can promote the degradation of tumor signal pathway proteins, such as ALK (related to the proliferation and survival of tumor cells), and provides a treatment strategy for crizotinib-resistant patients without secondary mutation. A series of combinations of HSP90 inhibitors with selective ALK inhibitors are in clinical research (NCT01712217 and NCT 01579994).
17-AAG (17-allylamine-17-demethoxygeldanamycin) is a potent HSP90 inhibitor obtained by structural modification of geldanamycin, which is also called tanespimycin. 17-AAG has obvious inhibition effect on the proliferation of human lung cancer A549 cell strain. 17-AAG can inhibit phosphorylation of active tyrosine kinase (RTK) receptors in EGFR-TKI resistant T790M cell strains, down-regulate EGFR and other RTK receptors, inhibit mammalian rapamycin target signaling, cause degradation of cell survival shunt activation protein, reduce activation of Ataxia telangiectasia Ataxia multiple gene (ATM), correspondingly block DNA excision repair mechanism and activation of key enzymes at multiple levels, and can promote the recovery sensitivity of lung cancer cell strains which have resistance to gefitinib and erlotinib. 17-AAG can increase the apoptosis effect of the anticancer drug, lead to the degradation of Epidermal Growth Factor Receptor (EGFR), block downstream signaling, and play a synergistic role of chemotherapeutic drugs, such as being capable of enhancing the antiproliferative effect of paclitaxel in lung cancer H3255(L858R EGFR) cell line.
The crizotinib and 17-AAG belong to insoluble drugs in water, the water solubility is poor (about 10-20 mu M), and different surfactants or organic solvents used for solubilization in the prior art have outstanding toxicity problems, so that great challenges are brought to the design and preparation of an anti-cancer drug delivery system. In addition, if two drugs are used in sequence or simply superposed, because the two drugs have different pharmacokinetic characteristics in vivo, the concentration and the proportion of the drugs with mutual synergistic effect are difficult to achieve at the tumor part, and the challenge is particularly outstanding for the combined drugs to achieve the best effect. Therefore, the related drug delivery system research at present aims to replace the existing organic solubilizing agent with a new delivery medium without toxic and side effects, improve the solubility of the insoluble antitumor drug, load two or more drugs to be combined into the drug delivery system at the same time, and aim to achieve improved pharmacokinetic characteristics (namely, the drugs used in combination reach the tumor part at the same time and are in accordance with the drug concentration and proportion with synergistic effect), and try to overcome the drug resistance of multiple drugs and increase the distribution and concentration of the drugs at the tumor part, thereby reducing the distribution of the drugs in normal tissues and reducing the toxicity. Researchers at home and abroad have made many studies on new drug delivery systems, such as polymer conjugates; liposome nanoparticles; and block polymer micelles, etc., to increase water solubility, but there are few varieties that can be industrially produced and meet safety evaluation at present. Wherein the nano micelle preparation becomes a research hotspot in the last two decades. The drug can improve the solubility of the drug and control the drug release, and can be passively targeted to a tumor part through EPR effect (namely, the high permeability and retention effect of solid tumor is less than enhanced permeability and retention effect), which means that compared with normal tissues, molecules or particles with certain sizes tend to be more accumulated in the tumor tissues.
The existing nano micelle preparation mainly uses amphiphilic polymer, and has high drug-loading rate and good stability. The amphiphilic polymer is a block copolymer and comprises a hydrophilic block and a lipophilic block, the formed nano micelle is of a shell-core structure, the hydrophilic block is a shell on the surface and can be dispersed in water and physiological saline, and the lipophilic block forms the core of the micelle and is used for loading hydrophobic drugs. The most common hydrophilic block at present is polyethylene glycol (PEG), which has good water solubility and low toxicity. However, in 25% of patients, anti-PEG antibodies (due to the large amount of PEG contained in other daily chemical products such as shampoo and some foods) are already present, and may be generated after multiple administrations, thereby limiting the clinical application of PEG-based polymer micelles. Most of the lipophilic blocks in the amphoteric polymers are polymers widely used in FDA-approved drugs and medical materials (e.g., polylactic-co-glycolic acid (PLGA), polylactic acid (PLA), etc.).
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a crizotinib and 17-AAG composite polymer nano micelle injection.
The amphiphilic polymer adopts poly (2-methyl-2-oxazoline) or poly (2-ethyl-2-oxazoline) as a hydrophilic block to replace PEG, uses PLA as a lipophilic block, uses polymer nano-micelle as a common carrier of crizotinib and 17-AAG, provides a polymer nano-micelle injection capable of resisting the drug resistance of crizotinib for clinic, can greatly increase the water solubility and the preparation stability of the crizotinib and 17-AAG, prolongs the circulation time of the crizotinib and 17-AAG in blood, eliminates the toxic and side effects of a solubilizer in the existing preparation, and by the characteristics of the composite micelle, the two medicines are kept to be passively targeted to the tumor part in a fixed proportion through an EPR effect, so that a synergistic anti-tumor effect is generated, and the drug resistance which is possibly generated during the treatment of crizotinib is resisted. In addition, the polymer micelle can overcome the problems of anti-PEG antibody, antibody generation after multiple times of administration and the like, and has great clinical significance.
The technical scheme of the invention is as follows:
the crizotinib and 17-AAG composite polymer nano micelle injection with the synergistic effect comprises 1 weight part of crizotinib, 0.5-3 weight parts of 17-AAG and 10-100 weight parts of nano micelle polymer; the nano micelle polymer is poly (2-ethyl-2-oxazoline) -polylactic acid, namely PEOZ-PLA, or poly (2-methyl-2-oxazoline) -polylactic acid, namely PMOZ-PLA.
In the poly (2-ethyl-2-oxazoline) -polylactic acid, namely PEOZ-PLA, the polymerization degree of the poly (2-ethyl-2-oxazoline) PEOZ is 20-60 units, the average molecular weight is 1700-5100 Da, the polymerization degree of the polylactic acid PLA is 30-115 units, and the average molecular weight is 850-3300 Da.
In the poly (2-methyl-2-oxazoline) -polylactic acid, namely PMOZ-PLA, the polymerization degree of the poly (2-methyl-2-oxazoline) PMOZ is 20-60 units, the average molecular weight is 1700-5100 Da, the polymerization degree of the polylactic acid PLA is 30-115 units, and the average molecular weight is 850-3300 Da.
Preferably, the first and second electrodes are formed of a metal,
the preparation comprises 1 part by weight of crizotinib, 1 part by weight of 17-AAG and 10 parts by weight of nano micelle polymer; the nano-micelle polymer is poly (2-methyl-2-oxazoline) -polylactic acid, namely PMOZ-PLA, wherein the polymerization degree of the poly (2-methyl-2-oxazoline) PMOZ is 50 units, the average molecular weight is 4400Da, the polymerization degree of the polylactic acid PLA is 115 units, and the average molecular weight is 3300 Da.
The average particle size of the crizotinib and 17-AAG composite polymer nano micelle injection is 20-200 nm.
The average particle size of the crizotinib and 17-AAG composite polymer nano micelle injection is 30-100 nm.
Preferably, the first and second electrodes are formed of a metal,
the average particle size of the crizotinib and 17-AAG composite polymer nano micelle injection is 35 nm.
The preparation method of the injection comprises the following steps:
1) respectively dissolving the crizotinib, the 17-AAG and the nano micelle polymer in ethanol or acetonitrile to prepare stock solutions, then weighing the crizotinib, the 17-AAG and the nano micelle polymer stock solutions according to a prescription, mixing, oscillating and uniformly mixing;
2) injecting the obtained crizotinib, 17-AAG and nano micelle polymer mixed organic phase into a round-bottom flask or other sample bottles, slowly volatilizing the organic solvent to be completely dry under reduced pressure or normal pressure in a slight heating process, and forming a mixed thin layer film of the medicine and the nano micelle polymer material at the bottom of the bottle;
3) heating to 35-50 ℃, adding distilled water or water for injection, and slightly oscillating until the thin layer film is completely dissolved to form a clear solution;
4) and (3) cooling the clear solution to room temperature, centrifuging, removing the crizotinib and 17-AAG which are not loaded in the micelle by centrifugation, taking supernatant, and freeze-drying to obtain the crizotinib and 17-AAG composite polymer nano-micelle injection freeze-dried powder.
Preferably, the first and second electrodes are formed of a metal,
in the step 3), the temperature is increased to 45 ℃.
In the step 4), a protective agent is added during freeze drying, the freeze-drying protective agent is selected from any one or a mixture of mannitol, sucrose, lactose, trehalose, maltose and glucose, and the dosage of the freeze-drying protective agent is 5-10g/100ml crizotinib and 17-AAG composite polymer nano-micelle. The freeze-drying protective agent is added to increase the stability of the crizotinib and 17-AAG composite polymer micelle injection and prolong the validity period of the crizotinib and 17-AAG composite polymer micelle injection.
The crizotinib and 17-AAG composite polymer nano micelle injection is applied to preparation of a medicine for treating tumors, wherein the tumors are ALK positive non-small cell lung cancer and drug-resistant ALK positive non-small cell lung cancer medicines.
The invention has the following technical effects:
1) the preparation method of the crizotinib and 17-AAG composite polymer nano micelle provided by the invention is simple, easy to operate and good in repeatability, and can realize industrial large-scale production.
2) The invention provides an application of the crizotinib and 17-AAG composite polymer nano-micelle injection in treating ALK positive non-small cell lung cancer, drug-resistant ALK positive non-small cell lung cancer, patients with anti-PEG antibodies and patients generating PEG antibodies after multiple drug administrations, wherein preferably, the tumors are ALK positive non-small cell lung cancer, drug-resistant ALK positive non-small cell lung cancer and the like.
Compared with the existing crizotinib preparation, the crizotinib and 17-AAG composite polymer nano micelle provided by the invention has the following advantages:
1. the polymer material has good biocompatibility and high safety, and does not generate toxic and side effects such as anaphylactic reaction, body fluid shrinkage and the like caused by a solubilizer.
2. According to the invention, the crizotinib and the 17-AAG are simultaneously encapsulated in the polymer nano micelle, so that the water solubility and the stability of the crizotinib and the 17-AAG are improved, and the effective period of the crizotinib and the 17-AAG is prolonged.
3. The crizotinib and 17-AAG composite polymer nano micelle provided by the invention has smaller particle size, can effectively penetrate through tumor blood vessels, is gathered at tumor parts through an EPR effect, realizes a passive targeting effect, and thus improves the treatment effect of the medicament.
4. The crizotinib and 17-AAG polymer nano micelle injection provided by the invention can greatly prolong the circulation time of the crizotinib and 17-AAG in blood and reduce the toxicity of the crizotinib and 17-AAG.
5. The amphiphilic polymer related to the crizotinib and 17-AAG composite polymer nano micelle injection provided by the invention adopts poly (2-methyl-2-oxazoline) or poly (2-ethyl-2-oxazoline) as a hydrophilic block to replace PEG, has the hydrophilicity equivalent to that of PEG, has similar 'stealth' effect as PEG, and is used as a shell layer of the micelle, so that not only can the inner core and the carried medicine of the micelle be protected, but also the phagocytosis of mononuclear macrophages can be prevented.
6. The crizotinib and 17-AAG composite polymer nano-micelle injection provided by the invention can overcome the condition that a polymer nano-micelle preparation taking polyethylene glycol (PEG) as a shell is not suitable for use (for example, treating a patient with an antibody to the PEG or a patient who produces an anti-PEG antibody by multiple times of administration).
7. The crizotinib and 17-AAG composite polymer nano micelle injection provided by the invention can load crizotinib and 17-AAG in a polymer nano micelle according to a preferable proportion, and the release speed of the composition is controllable.
8. The crizotinib and 17-AAG composite polymer nano micelle injection provided by the invention can be used for remarkably improving the tissue distribution and the pharmacokinetic characteristics of the crizotinib and 17-AAG by controllably releasing the crizotinib and 17-AAG with a synergistic effect ratio at a tumor part, and reducing the cytotoxicity to non-tumor tissues.
9. The crizotinib and 17-AAG composite polymer nano micelle injection provided by the invention achieves the concentration and proportion of a synergistic effect in tumor cells, has a better tumor inhibition synergistic index (CI), and can greatly improve the anti-tumor curative effect compared with the anti-tumor curative effect of 17-AAG or crizotinib single drug.
10. The crizotinib and 17-AAG composite polymer nano micelle injection provided by the invention has a curative effect in drug-resistant ALK positive non-small cell lung cancer cells.
11. The preparation method of the crizotinib and 17-AAG composite polymer nano micelle injection provided by the invention is simple and easy to implement, wide in material source, low in cost, stable in product quality and convenient for realizing industrial production.
Drawings
FIG. 1 is an Atomic Force Microscope (AFM) spectrum of a composite polymer nano-micelle of crizotinib and 17-AAG prepared in the example of the present invention.
Fig. 2 is a particle size distribution diagram of a crizotinib and 17-AAG composite polymer nano-micelle injection freeze-dried powder redispersion prepared in an embodiment of the invention.
FIG. 3 is a graph showing the external release curve of the freeze-dried powder dispersion liquid of the crizotinib and 17-AAG composite polymer nano-micelle injection prepared in the embodiment of the present invention.
FIG. 4 shows the inhibitory effect of crizotinib and 17-AAG composite polymer nano-micelle injection prepared in the embodiment of the invention on drug-resistant ALK positive non-small cell lung cancer cells.
Fig. 5 shows the synergistic effect index (preferable drug ratio) of crizotinib and 17-AAG composite polymer nano-micelle injection prepared according to the embodiment of the present invention in drug-resistant ALK-positive non-small cell lung cancer cells.
Detailed Description
The hydroxyl polyoxazoline PEOZ-OH or PMOZ-OH is prepared by reacting monomer 2-ethyl-2-oxazoline (or 2-methyl-2-oxazoline) with cation ring-opening polymerization at 100 ℃ for 24 hours, and the initiator is 4-methyl tosylate (MeOTs). The crude product is precipitated with ethyl ether after passing through silica gel, and the obtained PEOZ-OH or PMOZ-OH and L-lactic acid are further polymerized for 24 hours in chlorobenzene solvent under the catalysis of stannous octoate at 140 ℃. The resulting PEOZ-PLA (or PMOZ-PLA) was purified by ether precipitation and the molecular weight and distribution were determined by Gel Permeation Chromatography (GPC). N, N-Dimethylformamide (DMF) as a mobile phase at a flow rate of 1.0 m/l/min; the GPC column was prepared from 5 μm Phenogel and passed through polymethyl methacrylate standards (available from Polymer Laboratories). The composition of the obtained polymer was confirmed by nuclear magnetic resonance (1H-NMR) detection in a deuterated chloroform solvent.
The present invention is further illustrated by the following examples, which are not intended to limit the invention in any way.
Example 1
In the invention, the preparation of the crizotinib and 17-AAG composite polymer nano micelle comprises the following steps:
1) weighing crizotinib and 17-AAG, respectively taking 10mg of crizotinib and 17-AAG, adding 1ml of absolute ethyl alcohol, and ultrasonically dissolving; weighing 100mg of PMOZ-PLA polymer, adding 10ml of absolute ethyl alcohol, and ultrasonically dissolving; crizotinib, 17-AAG and PMOZ-PLA polymer were mixed. The polymerization degree of PMOZ is 50 units, and the average molecular weight is 4400 Da. The polymerization degree of polylactic acid PLA is 115 units, and the average molecular weight is 3300 Da.
2) The mixture was added to a 50ml round bottom flask, the solvent was slowly volatilized under reduced pressure at 40 ℃ and vacuum was applied to remove the solvent residue, forming a mixed thin layer film of drug and nanomicelle polymer material (drug + PMOZ-PLA polymer) at the bottom of the round bottom flask.
3) Adding 10ml of distilled water at 45 ℃, and slightly shaking until the thin layer membrane is completely dissolved to form a clear solution;
4) and (3) cooling the clear solution to room temperature, centrifuging at 5000rpm for 10 minutes, removing the crizotinib or 17-AAG which is not loaded in the micelle by centrifugation, taking supernatant, and freeze-drying to obtain the crizotinib and 17-AAG composite polymer nano-micelle injection freeze-dried powder.
And (3) taking the crizotinib and 17-AAG composite polymer nano micelle freeze-dried powder prepared in the example 1 to measure the encapsulation efficiency.
Chromatographic conditions are as follows: octadecylsilane bonded silica gel as filler [ e.g., Inertsil ODS3(5 μm, 150 mm. times.4.6 mm), YMC Pack Pro C18(5 μm, 150 mm. times.4.6 mm) or other equivalent chromatographic columns](ii) a Gradient elution was performed according to table 1 using phosphoric acid-ammonium hydroxide-water (1.5:1.0:1000) (pH adjusted to 2.5 with phosphoric acid or ammonium hydroxide) as mobile phase a and acetonitrile as mobile phase B; the flow rate is 1.0 ml/min; the detection wavelengths are 210 nm and 333nm respectively; the amount of the sample was 20. mu.l.
TABLE 1 gradient elution procedure
The crizotinib and 17-AAG composite polymer nano micelle freeze-dried powder prepared in example 1 is taken, distilled water is added to be re-dispersed to the crizotinib concentration of about 1mg/ml, 20 mu 1 of dispersion liquid is taken, 180 mu 1 of acetonitrile is added to be fully dissolved by oscillation, then centrifugation is carried out, and 20 mu 1 of supernatant is taken to be injected by HPLC. And preparing acetonitrile standard solutions with the concentrations of 10, 20, 50, 100 and 200 mu g/ml from standard crizotinib or 17-AAG stock solutions, respectively sampling 20 mu l of the acetonitrile standard solutions by HPLC, and performing curve regression on peak areas obtained under various concentrations. The encapsulation efficiency of the crizotinib and 17-AAG composite polymer nano-micelle is calculated to be about 88.1 percent and 91.1 percent through a standard curve and the concentration of the crizotinib or 17-AAG medicament in the nano-micelle freeze-dried powder redispersion liquid.
Taking crizotinib and 17-AAG composite polymer nano-micelle freeze-dried powder prepared in example 1, adding distilled water to redisperse to a crizotinib concentration of about 0.1mg/ml, redispersing a drop of crizotinib and 17-AAG composite polymer nano-micelle freeze-dried powder which is further diluted by 500 times to be dropped on the surface of a newly peeled mica cathode, standing for about 2 minutes, removing redundant dispersion liquid, washing with double distilled water and drying in an argon environment (see figure 1). The atomic force microscope result shows that the nano micelle is a uniform spherical structure and has the particle size of about 20-30 nm. The particle size of micelle is measured in a Malvern nanometer particle size analyzer by 0.1mg/ml crizotinib and 17-AAG composite polymer nanometer micelle freeze-dried powder redispersion liquid, the obtained average particle size is 35.1nm, and the dispersity is PDI (shown in figure 2) which is 0.112.
Precisely transferring 0.1ml of redispersion liquid of the crizotinib and 17-AAG composite polymer nano micelle freeze-dried powder prepared in the example 1, putting the redispersion liquid into a dialysis device (the molecular weight cutoff is 3500), putting the dialyzed solution into a beaker, and taking 1OOml of PBS buffer solution as a release medium; the temperature was set at 37 deg.C, the sample was taken after 0.5, 1, 2, 4, 6, 8, 12 hours, the concentration of the unreleased drug crizotinib and 17-AAG in the dialysis device was measured by HPLC, and the cumulative release rate and time of the drug was calculated to obtain an in vitro release profile, as shown in FIG. 3. The two drugs are released from the polymer micelle at comparable rates, with about 75% of the drug released in 24 hours.
0.1ml of the re-dispersion of the crizotinib and 17-AAG composite polymer nano-micelle freeze-dried powder prepared in example 1 was precisely transferred, diluted 10-fold in a cell culture medium to a series of concentrations, added to cells cultured in a 96-well plate (human non-small cell lung cancer NCI-H3122 crizotinib-resistant cell strain, 4000 cells/well), after 24 hours of culture, the drug-containing medium was removed and a new medium was added for further culture for 72 hours, then MTT reagent (100. mu.g/well) was added for further culture at 37 ℃ for 3 hours, the medium containing MTT was removed, 100. mu.l of DMSO was added and data were read by a plate reader at 562 nm wavelength (SpectraMax M5, Molecular Devices plate reader). The cell survival rate is calculated by the number of cells which are not added with drugs and is compared with the control group, and the average half inhibition concentration (IC50) is obtained by a curve of the survival rate and the cell concentration; the same procedure added each drug alone to the cells to determine IC50 (fig. 4). The combination significantly reduced IC50 in NCI-H3122 crizotinib-resistant cells compared to the single agent.
Synergy index (CI) analysis was based on the Chou and Talalay method and calculated using CompuSyn software. Briefly, for the case of dual drug combination, the index of synergistic effect at each degree of inhibition (Fa) can be calculated by the following equation: CI ═ D)1/(Dx)1+ (D)2/(Dx)2, where (D)1 and (D)2 are the concentrations of each drug in the cocktail required to achieve a specific degree of Fa cytostatic; and (Dx)1 and (Dx)2 are concentrations required to produce a degree of cell inhibition by Fa with a single drug. The synergy index CI is then plotted against the degree of inhibition. In general, CI obtained between Fa-0.2 and Fa-0.8 is considered effective, and CI at IC50 (i.e., Fa-0.5) is used to evaluate whether a drug combination has strong synergistic effects at different ratios. If the CI value is less than 1, the synergistic effect is present, and if it is greater than 1, the counteracting effect is present, and if it is equal to 1, the drug combination is regarded as having no synergistic effect, and only a simple addition is present (fig. 5). FIG. 5 shows a synergy index of 0.32 at IC50, indicating that the crizotinib and 17-AAG composite polymer nanomicelle of example 1 has a strong synergistic effect of inhibiting the growth of tumor cells (NCI-H3122 crizotinib resistant cell line).
Claims (9)
1. A crizotinib and 17-AAG composite polymer nano micelle injection with synergistic effect is characterized in that: the preparation comprises 1 weight part of crizotinib, 0.5-3 weight parts of 17-AAG and 10-100 weight parts of nano micelle polymer;
the nano micelle polymer is poly (2-ethyl-2-oxazoline) -polylactic acid, namely PEOZ-PLA, or poly (2-methyl-2-oxazoline) -polylactic acid, namely PMOZ-PLA;
in the poly (2-ethyl-2-oxazoline) -polylactic acid, namely PEOZ-PLA, the polymerization degree of the poly (2-ethyl-2-oxazoline) PEOZ is 20-60 units, the average molecular weight is 1700-5100 Da, the polymerization degree of the polylactic acid PLA is 30-115 units, and the average molecular weight is 850-3300 Da;
in the poly (2-methyl-2-oxazoline) -polylactic acid, namely PMOZ-PLA, the polymerization degree of the poly (2-methyl-2-oxazoline) PMOZ is 20-60 units, the average molecular weight is 1700-5100 Da, the polymerization degree of the polylactic acid PLA is 30-115 units, and the average molecular weight is 850-3300 Da.
2. The injection according to claim 1, characterized in that: the preparation comprises 1 part by weight of crizotinib, 1 part by weight of 17-AAG and 10 parts by weight of nano micelle polymer; the nano-micelle polymer is poly (2-methyl-2-oxazoline) -polylactic acid, namely PMOZ-PLA, wherein the polymerization degree of the poly (2-methyl-2-oxazoline) PMOZ is 50 units, the average molecular weight is 4400Da, the polymerization degree of the polylactic acid PLA is 115 units, and the average molecular weight is 3300 Da.
3. The injection according to claim 1, characterized in that: the average particle size of the crizotinib and 17-AAG composite polymer nano micelle injection is 20-200 nm.
4. An injection according to claim 3, characterized in that: the average particle size of the crizotinib and 17-AAG composite polymer nano micelle injection is 30-100 nm.
5. The injection according to claim 4, characterized in that: the average particle size of the crizotinib and 17-AAG composite polymer nano micelle injection is 35 nm.
6. A process for the preparation of an injection according to any one of claims 1 to 5, characterized in that: the preparation method comprises the following steps:
1) respectively dissolving the crizotinib, the 17-AAG and the nano micelle polymer in ethanol or acetonitrile to prepare stock solutions, then weighing the crizotinib, the 17-AAG and the nano micelle polymer stock solutions according to a prescription, mixing, oscillating and uniformly mixing;
2) injecting the obtained crizotinib, 17-AAG and nano micelle polymer mixed organic phase into a round-bottom flask or other sample bottles, slowly volatilizing the organic solvent to be completely dry under reduced pressure or normal pressure in a slight heating process, and forming a mixed thin layer film of the medicine and the nano micelle polymer material at the bottom of the bottle;
3) heating to 35-50 ℃, adding distilled water or water for injection, and slightly oscillating until the thin layer film is completely dissolved to form a clear solution;
4) and (3) cooling the clear solution to room temperature, centrifuging, removing the crizotinib and 17-AAG which are not loaded in the micelle by centrifugation, taking supernatant, and freeze-drying to obtain the crizotinib and 17-AAG composite polymer nano-micelle injection freeze-dried powder.
7. The method of claim 6, wherein: in the step 3), the temperature is increased to 45 ℃.
8. The method of claim 6, wherein: in the step 4), a protective agent is added during freeze drying, the freeze-drying protective agent is selected from any one or a mixture of mannitol, sucrose, lactose, trehalose, maltose and glucose, and the dosage of the freeze-drying protective agent is 5-10g/100ml crizotinib and 17-AAG composite polymer nano-micelle.
9. The use of the crizotinib and 17-AAG composite polymer nano-micelle injection according to any one of claims 1 to 5 for the preparation of a medicament for treating tumors, wherein: the tumor is ALK positive non-small cell lung cancer and drug-resistant ALK positive non-small cell lung cancer drugs.
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